JP2005526956A - Method for detecting half-antibodies using chip-based gel electrophoresis - Google Patents

Abstract

The present invention provides a method of determining the presence of a polypeptide having a selected disulfide bond pattern using chip-based gel electrophoresis, for example, the polypeptide is a heterogeneously formed heterobicycle. A fully formed tetrameric antibody compared to a half-mer antibody. The invention further features a kit, which includes a chip and instructions for performing the method described above. The methods and kits of the invention can be modified for high-throughput applications for production of recombinant therapeutic antibodies and quality control monitoring.

Description

(Related information)
This application is filed on US Provisional Patent Application No. 60 / 393,038 filed on June 28, 2002 and US Provisional Patent Application No. 60 / 341,938 filed on December 19, 2001. The entire contents of both of which are hereby incorporated by reference.

  The contents of any patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entirety.

(Background of the Invention)
The immune response is a mechanism by which the body defends itself against foreign substances that invade it and cause infection or disease. This mechanism is based on the discrimination and binding of these foreign substances by antibodies. Once the substance is bound by the antibody, it becomes a target for destruction.

  An antibody is composed of four polypeptides (L: H: H: L), two light chains and two heavy chains. Most antibodies contain a disulfide bond between the four polypeptide chains. Often so-called half-antibodies are generated, in which disulfide bonds are not formed between heavy chain polypeptides.

  In some antibodies, such as the IgG4 class, 25-30% IgG antibodies are one heavy chain and one light chain, whether these molecules are recombinant or produced naturally. Is produced as a half antibody. For other antibody isotypes and subisotypes, half-antibody formation is associated with abnormal protein forms. For example, half-antibody formation can result from the structure of the hinge region, as in IgG4 antibodies, or is a deletion in the heavy chain constant domain, as in antibodies produced by certain myeloma.

  Half-antibodies do not have clear clinical symptoms, but they have been identified in the serum and urine of patients with various diseases such as multiple myeloma, plasma cell leukemia, and plasmacytoma Yes. To some extent half antibodies are also produced by murine hybridomas and myeloma and are a byproduct of recombinant antibody production in both animal and bacterial cells. Many of these antibodies are potentially less biologically active when incomplete and therefore have the ability to weaken the therapeutic efficacy of pharmaceutical preparations containing such half antibodies. . Therefore, the ability to detect such molecules is desired.

  A typical method for determining the amount of half-antibody in a sample is by standard gel electrophoresis. This method is prone to delay and error.

  Thus, there is a need for an improved method of determining the presence of half-antibodies (when compared to fully formed antibodies) in order to monitor antibody-based drugs and improve their quality control. doing.

(Summary of the Invention)
The present invention provides the aforementioned method of standard gel electrophoresis by providing an improved method for detecting the presence of a selected disulfide linked polypeptide (eg, a half-antibody) compared to a fully formed antibody. Solve a problem. Typically, a fully formed antibody is a tetramer comprising two heavy chain polypeptides and two light chain polypeptides joined by disulfide bonds (L: H: H: L), but half antibodies Is a disulfide-linked light chain polypeptide and a heavy chain polypeptide (L: H) that are incompletely disulfide bonded or not fully bonded to the corresponding light chain polypeptide and heavy chain polypeptide (H: L). (FIG. 11). Other exemplary molecules that are amenable to the methods described herein include any multimeric polypeptide having one or more disulfide bonds. The methods of the invention are particularly useful for characterizing disulfide-linked multimeric polypeptides that are intended for use in human therapeutic applications, as well as in diagnostic and research applications.

  For example, the method can include a polypeptide that is incompletely formed, i.e., a polypeptide having an insufficient number of disulfide bonds, as compared to a polypeptide having a desired number or configuration of disulfide bonds. Use chip-based gel electrophoresis, which can be easily and quickly identified. Two prominent examples include antibodies (eg, IgG4) that contain four polypeptide chains held together by disulfide bonds, and multi-chain ligands (eg, growth factors).

Accordingly, the present invention has several advantages including but not limited to:
An improvement that can quickly determine the amount of a selected disulfide-linked polypeptide (eg, fully formed antibody) compared to an incompletely bound polypeptide (eg, a half-antibody) or unwanted impurities A method for monitoring quality control during production (eg, recombinant production) of polypeptide multimers (eg, antibodies) for therapeutic use in human subjects, eg, And kits that can be used with commercially available chips for performing the methods of the invention. Accordingly, in one aspect, the invention provides for detecting the presence of an IgG4 polypeptide having a selected disulfide bond pattern in a sample. Provide a method. Here, a sample containing a polypeptide having a selected disulfide bond pattern is a channel having a separation medium effective to act as an obstacle to migration of the polypeptide having a selected disulfide bond pattern, and an electric field Is loaded onto a chip with at least two electrodes placed in the channel. An electric field is applied through the separation medium of the chip, thereby allowing the IgG4 polypeptide with a selected disulfide bond pattern to be compared to the separation of an IgG4 polypeptide without the selected disulfide bond pattern. Separation is achieved. The presence of an IgG4 polypeptide having a selected disulfide bond pattern is determined, for example, by the amount of fully formed (ie, disulfide bonded) antibody, half antibody (eg, percentage), or ratio thereof.

  In a related aspect, the present invention is directed to a sample comprising a mixture of polypeptide multimers having two or more polypeptide chains and comprising at least one disulfide bond between the polypeptide chains. A method for detecting the presence of a polypeptide having a disulfide bond pattern is provided. According to this method, a sample comprising a mixture of polypeptide multimers is placed in and within a channel having a separation medium effective to act as an obstacle to migration of a polypeptide having a selected disulfide bond pattern, Loaded on a chip with at least two electrodes to induce an electric field. An electric field is applied across the separation medium of the chip, thereby achieving separation of the polypeptide having a selected disulfide bond pattern compared to a polypeptide having no selected disulfide bond pattern. . The presence of a polypeptide having a selected disulfide bond pattern is detected. Such peptides are, for example, fully formed antibodies or half-antibodies.

  In certain embodiments, the methods are used to detect IgG4 half antibodies, IgG4 antibodies, the presence of polypeptide impurities, or the ratio of any of the foregoing antibodies or polypeptides.

  In related embodiments, the antibody is a monoclonal antibody (eg, a humanized antibody, particularly an anti-integrin antibody (eg, an anti-α-4-β-1 (VLA-4) antibody, anti-α-4-β)). -7 (VLA-7) antibody, or an antibody that binds to both VLA-4 and VLA-7 (eg, natalizumab))).

  In another embodiment, the sample contains a polypeptide (eg, a polypeptide having a disulfide bond (eg, a ligand for an antibody)) from about 1 μg / ml to about 500 μg / ml (any range of polypeptides therein). Concentrations (eg, 1-10 μg / ml; 10-100 μg / ml; 100-500 μg / ml; 500-1000 μg / ml; and 1000-5000 μg / ml and any overlapping or narrower ranges of the aforementioned polypeptide concentrations Range).

  In another embodiment, the method detects a polypeptide that is a ligand having a selected disulfide bond pattern (eg, a fully formed binding pattern compared to an incompletely formed binding pattern).

  In another embodiment, the method is suitable for detecting selected disulfide bonds in recombinantly produced polypeptides.

  In another embodiment, the method is suitable for detecting selected disulfide bonds in recombinantly produced polypeptides.

  In related embodiments, the method is suitable for detecting polypeptide impurities in the presence of the aforementioned ligands, antibodies, half-antibodies, or disulfide bonded polypeptides.

  In a related embodiment, the method detects selected disulfide bonds in polypeptides isolated from cell culture growth media.

  In a related embodiment, the separation medium used in the method is a gel polymer (eg, a non-reducing gel polymer).

  In related embodiments, the movement of the polypeptide in the separation medium is detected using a fluorescence detector.

  In related embodiments, the polypeptides are separated according to their molecular weight.

  In related embodiments, the separation mechanism includes isoelectric focusing, where molecules are separated based on their isoelectric point.

  In another embodiment, the tip used in the present separation method comprises a precast gel polymer.

  In yet another embodiment, the present invention provides a kit for detecting the presence of a polypeptide having a selected disulfide bond pattern. This kit is here for detecting the presence of chops and polypeptides having a selected disulfide pattern (eg, antibodies, half-antibodies, or polypeptide impurities or any proportion of the aforementioned antibodies or polypeptides). Instructions for carrying out the described method are provided.

  In yet another embodiment, the present invention provides a kit for determining the purity of a therapeutic polypeptide having a selected disulfide bond pattern. This kit provides instructions for determining the amount of a chip and a polypeptide having a selected disulfide bond pattern (eg, antibody, half-antibody, or polypeptide impurity or any proportion of the aforementioned antibody or polypeptide). Prepare.

  In a related embodiment, the invention features a kit for determining the presence or purity of a polypeptide having a selected disulfide bond pattern, the kit comprising a chip, a polypeptide having a selected disulfide bond pattern Instructions for performing the assay to determine the amount of, as well as one or more of the following components (e.g., separation medium, non-reducing buffer, protein dye, formulation buffer or separation medium) A method for inducing an electric field through.

  Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

(Detailed description of the invention)
In order to provide a clear understanding of the specification and claims, the following definitions are conveniently provided below.

(Definition)
As used herein, the term “polypeptide having a selected disulfide bond” means that the selected disulfide bond occurs between a light chain polypeptide and a heavy chain polypeptide. Includes half-antibodies that do not occur in between.

  The term “chip” includes any solid substrate having means for including a separation medium to which an electric field can be applied across the medium, so that the chip is a macromolecule (eg, a multimeric polypeptide (eg, Antibodies and half antibodies)) can be used to separate them.

  The term “separation medium” includes any compound (eg, a polymer gel) that can be used to differentially separate macromolecules (eg, multimeric polypeptides (eg, antibodies and half-antibodies)), and In general, it is understood to include a suitable buffer solution.

  The term “half antibody” includes an antibody in which there is no inter-heavy chain disulfide bond so that one light chain polypeptide and one heavy chain polypeptide correspond to one light chain polypeptide. Forms that do not bind to the chain polypeptide and one heavy chain polypeptide.

  The term “IgG4 class” includes a subclass of IgG immunoglobulins that are produced during a secondary immune response and are most commonly found in blood. These IgG antibodies typically comprise a γ4 heavy chain.

  The term “non-reduced” refers to conditions under which disulfide bonds (eg, disulfide bonds) are preserved. Specifically, it refers to the condition where the disulfide bond remains intact and is not converted to sulfhydryl.

  The term “isoelectric focusing” includes methods in which macromolecules move and converge with a pH gradient established by applying a charge to a solution of a carrier ampholyte according to their isoelectric point (pI).

  The term “integrin” includes any polypeptide representing a large family of transmembrane proteins, so named because it is involved in the adhesion of cells to the extracellular matrix. Accordingly, the term “anti-integrin antibody” is an antibody that binds to such a molecule. Examples of anti-integrin antibodies include anti-α-4-β-1 (VLA-4) antibodies, anti-α-4-β7 (VLA-7) antibodies, and antibodies that bind to both VLA-4 and VLA-7 (Eg, natalizumab (see, eg, US Pat. No. 6,033,665 and US Pat. No. 5,840,299)).

(Detailed explanation)
Methods have been developed to detect the presence of polypeptides (eg, antibodies) having selected disulfide bonds using chip-based gel electrophoresis. This method involves fully formed antibodies (eg, IgG4) compared to incompletely formed antibodies (eg, half antibodies lacking interchain disulfide bonds (which are disulfide bonded heterodimers)). Antibody), which is a disulfide-bonded tetramer, is particularly well-suited for detecting the presence, absence or relative amount (ratio). Furthermore, this method can be used to detect the presence of impurities (eg, polypeptide impurities) in a sample. This method typically uses the Agilent 2100 Bioanalyzer in combination with the Protein 200 (or 200 Plus) LabChip kit and Protein 200 assay software (available from Agilent Technologies), but with similarly adapted hardware, reagents, and software. Can be done.

  As proof of principle, the methods of the invention described herein use test samples (especially anti-integrin antibodies) obtained during the production of recombinant antibodies for therapeutic applications, It is understood that the method can be equally applied to any antibody sample whether it is naturally derived (eg, from serum), produced by a cell line (eg, a hybridoma), or produced in a transgenic organism. .

  This method offers several advantages over current SDS-PAGE methods, for example, chip-based gel electrophoresis is more highly automated and easy to use. In contrast to convenient SDS-PAGE, no additional manual staining or decolorization steps are required. In addition, automation can produce polypeptides at different stages during the production process, such as selection of clones from cell lines (eg, from a cell bank) through cell culture expansion and final production phase. Enables near real-time analysis.

  This method obtains a sample comprising a polypeptide having a selected disulfide linkage pattern, eg, a multimeric polypeptide (eg, antibody or half antibody) clustered by one or more disulfide bonds or linkages, and a separation medium Or by loading the sample with or contacting the sample with a gel (eg, an organic polymer, which is confined to an opening or channel on a solid substrate such as a chip). The separation medium or gel is then subjected to an electric field (ie, an electric current is applied to the gel), so that the polypeptide molecules move through the separation medium based on molecular weight. Movement of the polypeptide through the separation medium or gel is performed under conditions that preserve the disulfide linkage pattern (or disulfide bonds) of the polypeptide (eg, under non-reducing conditions). Thus, polypeptides having different disulfide linkage patterns can be distinguished, and in particular, multimeric polypeptides such as tetrameric antibodies can be distinguished from heterodimeric half antibodies.

  In one embodiment, the actual detection of the migration pattern of a polypeptide having a selected disulfide linkage is performed using a dye (eg, a fluorescent dye), which can interact with the polypeptide; And for example, it can be visually monitored using an optical detection device (eg, a fluorescence reader).

  Conditions for conducting gel electrophoresis, reagent concentrations, detection dyes and appropriate equipment are well known in the art. Furthermore, there are many variations of buffer conditions and detection reagents that can be adapted to the methods of the invention. Thus, the present invention has the advantage of being easily incorporated into established protocols that do not require extensive reoptimization. Furthermore, the methods of the invention can be used to monitor the nature of any polypeptide having a disulfide linkage.

  In one particular embodiment, the method of the present invention can be used as described above for Protein 200 (or 200 Plus) LabChip Kit and dedicated Protein 200 assay software (which can up to 10 sample polypeptide sizes within 45 minutes). And provide detailed data on concentrations) and using an Agilent 2100 Bioanalyzer. Software calculations are performed, the molecular weight for each polypeptide detected in the sample (based on the amount of fluorescence detected over time), the relative polypeptide concentration and sample corresponding to each other polypeptide detected The amount (percentage) of each polypeptide compared to the total relative polypeptide concentration in is provided. This method allows for rapid measurement of the amount (percentage) of half-antibody molecules in a given sample without the need for further procedures necessary to perform SDS-PAGE. This method also allows detection of impurities (eg, polypeptide impurities).

  The data presented in the examples below indicate, among other things, the high accuracy of the method for measuring the amount of fully formed antibody, the relatively incompletely formed antibody (ie, half antibody) and impurities. Prove that. Furthermore, the method of the invention using a chip-based gel electrophoresis approach is an example of a power that incorporates multiple operations on a chip (eg, staining, separating and diluting protein samples). This provides an excellent approach to simple gel electrophoresis.

  The present invention also provides kits for convenient implementation of the methods of the present invention. In one embodiment, the present invention provides a kit for detecting the presence of a polypeptide having a selected disulfide bond pattern, which kit performs the chip and the methods described herein. Includes instructions for. The kit may also comprise at least one other component, such as a separation medium, a non-reducing buffer, a protein dye, a formulation buffer, and a means for inducing an electric field through the separation medium.

  In a preferred embodiment of the present invention, any of the above kits are conveniently used with a separation medium, non-reducing buffer, protein dye, chip, and / or means for inducing an electric field through the separation medium. Can be further designed, packaged or provided with instructions (eg, available from Agilent Technologies (see US Pat. No. 6,254,754)).

  Other commercially available separation media (gels), detection agents (dyes), detectors, chips, and devices for inducing an electric field are encompassed by the present invention to carry out the methods disclosed herein. (Eg, US Pat. Nos. 6,176,990; 6,261,430; 5,750,015; 5,449,446; and No. 5,427,663).

  The methods of the present invention are applicable to a variety of uses, including bioproduction, research, diagnostic applications, and forensic medicine.

(Biological production)
The methods and kits of the present invention have a variety of applications in the biological production of polypeptides having disulfide bonds. Indeed, antibodies (multimeric ligands) are a preferred class of therapeutic polypeptides produced commercially, which are multimeric polypeptides linked by disulfide bonds.

  Accordingly, the methods of the invention, as exemplified herein, can be used in any relevant stage of disulfide bond polypeptide bioproduction (eg, clonal selection from clone banks, cell culture growth, and small scale and Easily applied to monitoring or quality control (QC) of large scale production (eg using a biofermentor). Since the method of the present invention is rapid and can be automated, near real-time analysis can be performed during production and, if desired, to alter production parameters to affect the quality of product output. Can be used.

(Research application)
The methods and kits of the present invention have a variety of research applications. For example, they can be used in any research application for a limited amount of sample where the analysis must be performed quickly or contain a polypeptide having a disulfide bond (eg, a multimeric polypeptide such as an antibody or ligand). Useful for.

  Other applications of the method of the invention for research use will be readily apparent to those skilled in the art.

(Diagnostic application)
The methods and kits of the invention are useful in a variety of diagnostic applications, such as the detection of inappropriately bound polypeptides (eg, half antibodies) in a patient.

  The methods and kits of the invention described herein also detect antibodies and / or half-antibodies associated with a disease (eg, genetic disorder or cell disorder (eg, cancer)) or Can be used to characterize.

(Forensic application)
Forensic science relates to the scientific analysis of criminal evidence. Forensic biology applies molecular biology, biochemistry and genetics laboratory techniques to the testing of biological evidence, eg, for the purpose of positively identifying the perpetrators of crime. Typically, the sample size of such biological evidence (eg, blood) is small but still contains a sufficient amount of polypeptide (eg, antibody) that can be detected according to the methods of the invention.

  Thus, the improved chip-based gel electrophoresis technique of the present invention can be used to detect polypeptides (eg, to identify antibodies) from very small biological samples.

  The following examples are included for purposes of illustration and are not to be construed as limitations of the invention.

(Experiment)
Throughout the examples, the following materials and methods were used unless otherwise indicated.

(Materials and methods)
In general, the practice of the present invention uses conventional techniques of standard techniques in chemistry, molecular biology, recombinant DNA techniques, immunology (especially, immunoglobulin techniques) and electrophoresis, unless otherwise indicated. See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocol, Biosciences (Methods in Molecular S. 510). , Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Application Series, 169), McCafety ed., Ir Pr (1996); S. H. L. Press, Pub. (1999); Current Protocols in Molecular Biology, edited by Aubel et al., John Wiley & Sons (1992). Bousse et al., Protein Sizing on a Microchip, Anal. Chem. 73, 1207-1212 (2001); Knapp et al., Commerarized and Emerging Lab-on-a-Chip Applications, Processeds of the μTAS 2001 Symposium, Ramsey, J. Am. M.M. & Van den Berg, A.A. , 7-10 (2001); and Mhatre et al., Strategies for locating disbonded bonds in a monoclonal antigenic via mass spectrometry, Rapid Commun. See Mass Spectrom, 13 (24) 2503-2510 (1999).

(Equipment and sample)
Separation and detection of antibody samples was performed using an Agilent 2100 Bioanalyzer instrument in conjunction with the Protein 200 (or 200 Plus) LabChip kit and dedicated Protein 200 assay software. The Bioanalyzer uses epifluorescent detection with a 10-mW semiconductor laser emitting at 630 nm. The instrument also includes 16 independently programmable high voltage sources.

（サンプル調製）
タンパク質サンプルを、処方緩衝液（例えば、ＰＢＳ、または１０ｍＭのリン酸ナトリウム、ｐＨ６．０、１４０ｍＭのＮａＣｌ、０．０２％のＴｗｅｅｎ−８０ＫＲ）を使用して必要に応じて希釈し、そして変性緩衝液と２：１の比で混合することによって変性し、そして５分間で１００℃まで加熱した。変性緩衝液は、Ａｇｉｌｅｎｔ Ｐｒｏｔｅｉｎ ２００ Ａｓｓａｙキットで提供され、４％のＳＤＳおよび２９０μｇ／ｍｌのミオシン内部マーカーを含む。変性後、実験のためのサンプルを、脱イオン化水中のＡｇｉｌｅｎｔ Ｔｅｃｈｎｏｌｏｇｉｅｓからの下側（ｌｏｗｅｒ）マーカー色素（ｄｙｅ）の１０％溶液（励起波長／発光波長 ６５０／６８０ｎｍ）で、１：１５に希釈した。最適なタンパク質濃度は、３０００μｇ／ｍｌであると決定された。標準的な技術を使用してＮＳＯ細胞中で組換え的に産生された抗インテグリン抗体の、１０個を超えるそれぞれのサンプルを、以下のアッセイ中で使用した。

(Sample preparation)
Protein samples are diluted as necessary using formulation buffer (eg, PBS or 10 mM sodium phosphate, pH 6.0, 140 mM NaCl, 0.02% Tween-80KR) and denaturing buffer Denaturation by mixing with liquid in a 2: 1 ratio and heating to 100 ° C. for 5 minutes. Denaturation buffer is provided with the Agilent Protein 200 Assay kit and contains 4% SDS and 290 μg / ml myosin internal marker. After denaturation, samples for experiments were diluted 1:15 with a 10% solution of lower marker dye (dye) from Agilent Technologies in deionized water (excitation wavelength / emission wavelength 650/680 nm). . The optimal protein concentration was determined to be 3000 μg / ml. Over 10 individual samples of anti-integrin antibodies recombinantly produced in NSO cells using standard techniques were used in the following assays.

(Chip preparation (chip priming))
For all separation experiments, every channel in the glass chip is loaded into the upper right well labeled “G” with a sieving matrix and the Agilent Reagent kit guide (2000) (incorporated by reference). Loaded by applying pressure for 1 minute according to the standard procedure described in). The sieve matrix used is 3.25% polydimethylacrylamide-based polymer in Tris-Tricine buffer (pH 7.6) (120 mM Tricine, 42 mM Tris) and 0.25% SDS (8. 7 mM final concentration) and 4 μM lower marker (“Agilent” dye). This sieve matrix is prepared according to standard procedures using the reagents provided in the kit. All four wells labeled “G” were filled with this solution. SDS dilution wells (labeled “DS”) contain only sieve matrix and Tris-Tricine buffer. The protein sample (total volume 6 μl) is applied to all remaining wells on the chip except the bottom, G next well. This well (which is marked with a ladder mark) is loaded with a protein ladder containing seven proteins having a molecular weight of 14-210 kD.

(Data acquisition and analysis)
In the chip, electrophoresis continues to move from the well to the central channel to each sample. As the sample moves down to the central channel, these samples are sized and passed through a laser that ultimately excites the fluorescent dye that binds to the sample. Data acquisition and analysis is performed using an Agilent Technologies 2100 Bioanalyzer Software (version A.02.01). This software analysis data is based on fluorescence intensity versus time. Concentration quantification and protein sizing is accomplished by comparing against a sizing ladder and an internal standard (“marker”), which moves with each sample. This data includes data as gel-like images, electropherograms, as well as migration time, fluorescence intensity (as “Corr region”), size, relative concentration, absorption concentration (selection), and “total%” It is shown in tabular format (a table combining the results).

  The electropherogram from Agilent 2100 Bioanalyzer visualizes their separation according to the molecular weight (kD) of the protein. This Protein 200 ladder is electrophoresed from the designed ladder well onto each chip. After analysis of the Protein 200 ladder, the software creates a migration time calibration curve for the molecular weight of each protein in the ladder. This calibration curve is then used to determine the size of each of the proteins detected in the 10 samples. The lower and upper markers that are electrophoresed with each of the 10 samples correct for slight drifts in travel time and ensure accurate sizing. This software performs sizing automatically based on alignment with internal markers and external protein standards. The determination of molecular weight is based on the measured electrophoretic migration time (seconds).

  Relative protein concentrations (μg / ml) were determined based on measurements of peak areas and comparison of them with an internal standard of known protein concentration (upper marker). Total% is calculated based on the relative concentration.

  When detection is complete, the results for each sample are displayed in real time. The first result is acquired in 7 minutes, where each subsequent analysis follows at 2 minute intervals. These results are shown for each sample in tabular form, gel-like image and electropherogram.

  Each electropherogram includes a lower marker peak, a system peak, and an upper marker peak. The upper marker is 95% pure and contains two small impurities, 18 kD and 25 kD. This upper marker impurity level is corrected by software for concentration determination. Since these peaks are relatively low molecular weight, these peaks do not interfere with the antibody (or half-antibody) peaks.

(Example 1)
(Method for determining the accuracy and reproducibility of chip-based gel electrophoresis sizing)
Analysis of the anti-integrin antibody sample identified one major peak corresponding to the fully formed anti-integrin antibody (MW approximately 160 kD) and a small peak of approximately 89 kD corresponding to the half antibody. The same peak pattern was found for different anti-integrin antibody samples, which were also processed under non-reducing conditions. The molecular weight of these two peaks for different samples is shown in FIG. 2, and the same data for anti-integrin antibodies at different protein concentrations is shown in FIG. This data is very reproducible, but the molecular weight of the main peak decreases when the sample concentration increases from 50 to 4900 μg / ml, and its average MW is 161.1 for intact anti-integrin antibodies. The half antibody was 89.1.

  (Table 2. Sizing accuracy of chip-based method)

（実施例２）
（チップベースのゲル電気泳動とＳＤＳ−ＰＡＧＥとの比較）
抗インテグリンサンプル中に半抗体は、非還元ＳＤＳ−ＰＡＧＥによって、８０ｋＤまで移動するバンドとして検出可能である。従来のＳＤＳ−ＰＡＧＥにより得られたデータを、チップベースの方法によって得られたデータと比較した。非還元条件下のＳＤＳ−ＰＡＧＥを、標準的な技術に従って実施した（例えば、Ｍａｎｉａｔｉｓら（１９９５）；Ａｕｓｕｂｅｌら（２００１）を参照のこと）。代表的に、非還元条件下のＳＤＳ−ＰＡＧＥ分析について、通常、３μｇのタンパク質サンプルを、ゲルにロードする。チップベースの方法について、１μｇほどの少量で十分である。

(Example 2)
(Comparison between chip-based gel electrophoresis and SDS-PAGE)
The half antibody in the anti-integrin sample can be detected as a band that migrates to 80 kD by non-reducing SDS-PAGE. Data obtained by conventional SDS-PAGE was compared with data obtained by chip-based methods. SDS-PAGE under non-reducing conditions was performed according to standard techniques (see, eg, Maniatis et al. (1995); Ausubel et al. (2001)). Typically, for SDS-PAGE analysis under non-reducing conditions, typically 3 μg of protein sample is loaded onto the gel. For chip-based methods, as little as 1 μg is sufficient.

  Subsequently, SDS-PAGE analysis was performed on several antibody samples at the same low protein concentration (or loading protein) as for the chip-based method (Table 3 and Figure). 5). These chip-based results demonstrate a good correlation between these two methods. Data obtained for a wide variety of antibody samples showing a high proportion of half-antibodies are presented in Table 3. For example, both SDS-PAGE and chip-based methods detected a high proportion of half-antibodies in anti-integrin antibody samples 5, 8, 9 and 10 (Table 4). These results also demonstrate a good correlation between these two methods.

抗インテグリン抗体サンプル１を、異なる濃度で、チップベースの方法（半抗体の割合を、合わせた結果表から決定した）および従来のＳＤＳ−ＰＡＧＥによって分析した。

Anti-integrin antibody sample 1 was analyzed at different concentrations by a chip-based method (the proportion of half-antibodies was determined from the combined results table) and conventional SDS-PAGE.

示される濃度での異なる抗体サンプルを、チップベースの方法およびＳＤＳ−ＰＡＧＥ方法によって分析した。

Different antibody samples at the indicated concentrations were analyzed by chip-based and SDS-PAGE methods.

(Example 3)
(Method for determining the linear dynamic range of an assay)
This example provides a method that can be used to determine the range of protein concentrations for which the results from this assay are accurate. To determine the linear dynamic range of this assay, anti-integrin antibody samples at concentrations of 12.5-4900 μg / ml were analyzed. FIG. 2 (panels AD) shows a control sample (A) containing different anti-integrin antibodies and formulation buffer at the lowest concentrations (12.5 μg / ml, 50 μg / ml and 100 μg / ml) (B, C, D). The result of the analysis is shown.
The peak corresponding to this half-antibody band can only be detected at a total sample concentration of 50 μg / ml (C), whereas the intact antibody peak (MW about 171) can be easily seen at 12.5 μg / ml. (B). With the staining approach used for chip-based methods, it should be expected that staining saturation can be reached at somewhat higher concentrations of protein. An insufficient amount of dye can be shown to bind to the SDS-protein complex in a quantitative manner. As a result, the amount of protein in the sample is underestimated / overestimated. It is therefore important to identify the sample concentration that gives a linear correlation between the signal (fluorescence intensity / relative concentration) and the “true” sample concentration. The “true” sample concentration was determined by a standard procedure based on _A280 . These data are presented as total sample concentrations and as concentrations corresponding to antibodies and half antibodies (approximately 10%). The combined results table shows the different parameters of the assay results, including fluorescence intensity (as “Corr. Area”, relative sample concentration, and absolute sample concentration).

  Since the experiment showed that the correlation between true sample concentration and fluorescence intensity was low, the “relative sample concentration” was tested. Relative protein concentration (μg / ml) is obtained based on measuring peak areas and comparing these measurements against the upper marker (myosin) used as an internal standard for each sample. The Agilent 2100 Bioanalyzer software automatically determines the peak area of the unknown protein and the upper marker for each sample. The relative concentration of the unknown protein within a sample is then calculated by the software based on the known concentration of the upper marker. Inclusion of the upper marker in each sample calibrates for differences in sample injection into separate channels and allows reproducible quantification. The true sample concentration is compared (“X”) to the concentration (relative and absolute) determined by the software separately for anti-integrin and half antibodies. A reference standard sample (Sample 7) was diluted and tested as shown in FIGS. The reported sample concentration does not include a 15-fold dilution of the sample with water before loading the sample onto the chip.

This assay is linear over a two-digit scale, eg, from the lower detection limit (12.5 μg / ml) to 2000 μg / ml, based on the peak corresponding to the intact anti-integrin antibody (FIG. 4). This linearity decreases slightly but gradually decreases beyond 2000 μg / ml (FIG. 5). A correlation coefficient of R ² = 0.98 was determined based on the intact antibody peak for a sample concentration of 500-4900 μg / ml. R ² = 0.996 was observed at a concentration of 100-2000 μg / ml (FIG. 4).

(Example 4)
(Method for determining the linear dynamic range of the chip-based method)
The following example provides a method that can determine the dynamic range of a chip-based method. Anti-integrin antibody standards (Sample 7) were prepared at protein concentrations of 100 μg / ml, 200 μg / ml, 500 μg / ml, 1000 μg / ml, 1500 μg / ml, 2000 μg / ml, 3000 μg / ml, 4000 μg / ml, and 4900 μg / ml. analyzed. It was diluted stock solution having a concentration of 4900μg / ml (concentrations _were determined on the basis of the _{A 280).} Based on the combined results table (provided by the software; Table 4), the relative concentrations of the peaks corresponding to the intact antibody peak (Figures 3 and 4) and the half antibody peak (Figures 5 and 6). The range of protein concentrations was determined and a rough linear dynamic range was observed.

(Example 5)
(Method to determine correlation between determined absolute and related antibody and total antibody sample concentrations)
The following examples provide methods by which the actual and relative concentrations of protein samples can be correlated. Two sets of antibody standards (Sample 7) were analyzed at the following protein concentrations: 1) 12.5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 250 μg / ml, 500 μg / ml, 1000 μg / ml ml, 2000 μg / ml, and 4900 μg / ml and 2) 100 μg / ml, 200 μg / ml, 500 μg / ml, 1000 μg / ml, 1500 μg / ml, 2000 μg / ml, 3000 μg / ml, 4000 μg / ml, and 4900 μg / ml. The stock solution was diluted (concentration determined based on _A280 ). With the exception of 4900 μg / ml, all sample concentrations used a calibration curve for determination of absolute concentrations. Total relative and absolute concentrations were determined from the combined results table.

Similar graphs based on half-antibody peaks show a linear dynamic range for samples at concentrations of 50 μg / ml to 2000 μg / ml. A correlation coefficient of R ² = 0.949 was determined for sample concentrations from 100 μg / ml to 4900 μg / ml (FIG. 5). A correlation coefficient of R ² = 0.987 (FIG. 6) was observed at a concentration of 100 μg / ml to 2000 μg / ml. The lower concentration was not tested because preliminary data already indicated the absence of a half antibody peak at 10-20 μg / ml. FIG. 7 shows a good correlation between the relative and absolute concentrations of the sample determined by software analysis, and the actual sample concentration determined based on _A280 measurements. At low total sample concentrations, the percent (%) half antibody (relative concentration of half antibody) is lower than the% half antibody obtained at higher sample concentrations (linear range).

(Example 6)
(Method for determining optimal sample concentration)
The following examples provide a method for determining the optimal protein concentration that should be used to obtain optimal results. To determine the optimal sample concentration, the results for% half antibody obtained at different sample concentrations were analyzed (FIGS. 8 and 9). The% half antibody determined at low concentration is significantly lower (100 μg / ml or less). Based on these results, the recommended optimal sample concentration is 3000 μg / ml.

(Example 7)
(Method for determination of detection limit (LOD))
The following example provides a method for determining the lowest concentration at which half-antibodies are accurately detected. The detection limit was determined based on the observation that the lowest detectable concentration of half antibody was 2.7 μg / ml at a sample concentration of 50 μg / ml. This determined concentration that occurred with the low% half antibody detected (4.5% compared to 10% on average, see below), hence the actual concentration of half antibody in the sample is 2 Can be twice as high (about 5 μg / ml). Thus, the optimal sample concentration suggested for this assay is about 3000 μg / ml. Based on this assumption, the limit of quantification for% half antibody can be determined as half antibody concentrations of 5 μg / ml (2 ng loading) and 0.17% (5 μg / ml / 300 μg / ml). If the% half antibody is about 10%, this corresponds to a sample concentration of 50 μg / ml.

  This calculation was performed based on the relative concentration data. FIG. 10 shows the raw data at 50 μg / ml. The half antibody peak with MW 89.66 is clearly distinguished from the background.

(Example 8)
(Method for determining limit of quantification (LOQ))
The following example provides a method for determining the maximum concentration at which half-antibody determination is accurate. Specifically,% half antibodies determined at different sample concentrations were compared to determine quantitation limits. The LOQ is determined to be 613 μg / ml sample concentration and / or 1.9%. This was calculated based on the assumption that the optimal sample concentration was 3000 μg / ml and the mean% half antibody of the antibody reference standard was 9.1%. Linear regression was applied to the point (sample concentration) giving a% half antibody 20% higher or lower than the mean (7.3-11%).

Example 9
(Method for determining the reproducibility and accuracy of chip-based methods)
The following examples provide methods for testing the reproducibility and accuracy of chip-based assays. The reproducibility of the assay when using multiple chips or when using the same sample and a different chip was tested by using the same sample and the same chip. The antibody reference standard (Sample 7) was used to confirm the reproducibility of the chip-based method. Table 5 shows data obtained by running the same sample on the same chip. These data (% half antibody, “% total” determined from the combined results table) are STDEV = 0.5 and% STDEV = 0.5 and 6.0 for the antibody and half antibody, respectively. Very similar.

  (Table 5. Comparative data for different samples run on the same chip)

異なるチップ上での同じサンプルの泳動のデータ分析を、表６に示す。０．１、０．２、０．３、０．４、および０．５μｇで決定された％半抗体についての平均は９．５であり、ＳＴＤＥＶは０．３、および％ＳＴＤＥＶは３．３６である。

A data analysis of the migration of the same sample on different chips is shown in Table 6. The average for% half antibodies determined at 0.1, 0.2, 0.3, 0.4, and 0.5 μg is 9.5, STDEV is 0.3, and% STDEV is 3.36. It is.

  (Table 6. Comparative data for the same sample run on different chips)

抗体サンプル（すなわち、１２番）を、％半抗体および抗体を決定するために、０．１、０．２、０．３、０．４、および０．５μｇの量（濃度３７０、７５０、１１００、１５００、および１８７０μｇ／ｍｌ）でロードした。このアッセイは、多種のサンプル緩衝液および添加物を許容し得ることもまた観察された。

Antibody samples (i.e., # 12) are analyzed in 0.1, 0.2, 0.3, 0.4, and 0.5 μg amounts (concentrations 370, 750, 1100) to determine% half antibody and antibody. 1500, and 1870 μg / ml). It was also observed that this assay can tolerate a wide variety of sample buffers and additives.

(Example 10)
(Method for determining the reproducibility and accuracy of chip-based methods for detecting impurities)
The following examples provide methods for testing the reproducibility and accuracy of chip-based assays for detecting impurities in samples (eg, samples containing recombinant antibodies).

  The standard reproducible protein preparation process, wherein the reproducibility of this assay is detected, for example, by a specific concentration of a known marker protein added intentionally, or by other means (eg SDS-PAGE) and Tested by using several recombinant antibody samples with any residual impurities from the recovery process.

  In one experiment, three marker proteins (trypsin inhibitor (MW 21 kD), ovalbumin (MW 46 kD), and galactosidase (MW 116 kD)) were added to the recombinant antibody reference standard (ie, natalizumab), As a result, the final concentration of this sample was 4800 μg / ml, and the final concentration of marker protein corresponded to 0.75% impurity, 1% impurity, 1.2% impurity, and 1.5% impurity. Recovery data is shown in Table 7 and / or FIG.

  (Table 7. Collection of impurities in recombinant protein sample)

チップベースの方法の感度を決定するために、ＳＤＳ−ＰＡＧＥおよびチップベースの方法によって得られた不純物分析データの比較を行った（図１３および図１４）。還元ＳＤＳ−ＰＡＧＥは、チップベースの方法が、０．１％の検出限度（ＬＯＤ）、および０．５％の定量限度（ＬＯＱ）を有することを示した。

To determine the sensitivity of the chip-based method, impurity analysis data obtained by SDS-PAGE and the chip-based method were compared (FIGS. 13 and 14). Reduced SDS-PAGE showed that the chip-based method had a detection limit (LOD) of 0.1% and a quantification limit (LOQ) of 0.5%.

  Therefore, we concluded that chip-based methods are an effective and reliable approach for detecting and quantifying impurities in polypeptide samples (eg, samples containing recombinant proteins such as antibodies). .

(Equivalent)
For those skilled in the art using only routine experimentation, there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

FIG. 1 shows a chromatograph showing the method sizing accuracy (by migration time) as a function of sample concentration for intact antibodies (anti-integrin antibodies) and corresponding half antibodies. FIG. 2 shows a chromatograph showing the increased amount of antibodies (anti-integrin antibodies) compared to controls (panels AD) to determine the linear dynamic range of the assay. 3, using a concentration range of 100μg / ml~5000μg / ml, the antibody and the relative concentration (samples for accurate sample concentration (anti-integrin antibodies) _{(A 280} determined by reading), a known concentration were run Shows the linearity of the method by plotting. FIG. 4 shows the linearity of the method by plotting the concentration of antibody (anti-integrin antibody) relative to the sample concentration using a concentration range of 100 μg / ml to 2000 μg / ml. FIG. 5 shows the linearity of the method by plotting the relative concentration of the half antibody against the sample concentration, using a concentration range of 100 μg / ml to 5000 μg / ml. FIG. 6 shows the linearity of the method by plotting the relative concentration of the half antibody against the sample concentration using a concentration range of 100 μg / ml to 2000 μg / ml. FIG. 7 shows the correlation between the relative and absolute concentrations of the sample determined by the actual sample concentration based on software analysis and absorbance readings. FIG. 8 shows a graph showing the percentage of half antibody at sample protein amounts ranging from 0.1 μg to 0.5 μg. FIG. 9 shows a diagram showing the percent half-antibody of sample protein concentrations ranging from 10 μg / ml to 4900 μg / ml used to determine the optimal sample concentration. FIG. 10 shows the chromatograph used to determine the lowest detectable concentration of half-antibody using the above assay. FIG. 11 shows the structure of an antibody with the indicated intrachain and interchain disulfide bonds. Typically, an IgG4 intrachain disulfide bond occurs between light chain residues 23 and 92; between 138 and 98; between heavy chain residues 22 and 96; between 145 and 201; And 322; and between 368 and 426. Disulfide bonds between IgG4 heavy chains typically occur at residues 227 and 230. FIG. 12 shows a bar graph of an exemplary sample having a known concentration of “impurity” represented by a marker protein to determine the level of impurity detection using a chip-based gel electrophoresis method ( See also, for example, Table 7 and the description). FIG. 13 shows a digital image of an SDS-PAGE analysis of contaminants that can be detected using chip-based gel electrophoresis in an exemplary test sample and recombinant antibody preparation. The arrow pointing to the right indicates a known contaminating protein band (sample / lane 1 contains trypsin inhibitor, sample / lane 2 contains ovalbumin, sample / lane 3 contains β-galactosidase, and sample / lane Lane 4 shows control standards). The arrows pointing to the left indicate contaminating proteins from the recombinant sample, and “HC” and “LC” indicate the heavy and light chains of the recombinant antibody (ie, natalizumab), respectively. FIG. 14 shows a chromatograph using a chip-based method of a representative sample containing contaminating proteins previously tested by SDS-PAGE (FIG. 13). Panels represented from top to bottom correspond to lanes 1 to 4 of the gel image shown in FIG. In each case, the marker protein (ie trypsin inhibitor, top panel; ovalbumin, bottom 2 panel; β-galactosidase, bottom 3 panel) is easily detected as a peak (arrow (See), this does not occur in the control standard (bottom panel).

Claims (26)

A method for detecting the presence of an IgG4 polypeptide having a selected disulfide bond pattern in a sample, comprising:
Loading a sample comprising a polypeptide having a selected disulfide bond pattern onto a chip comprising a channel having a separation medium effective to act as an obstacle to migration of the polypeptide having a selected disulfide bond pattern And at least two electrodes are disposed in the channel to induce an electric field,
Applying an electric field across the separation medium of the chip, whereby the IgG4 poly having the selected disulfide bond pattern as compared to the IgG4 polypeptide not having the selected disulfide bond pattern Determining the presence of an IgG4 polypeptide having the selected disulfide bond pattern, wherein separation of the peptides is achieved, and
Including the method. A method for detecting the presence of a polypeptide having a selected disulfide bond pattern in a sample, the sample comprising a mixture of polypeptide multimers having two or more polypeptide chains, and Comprising at least one disulfide bond between the polypeptide chains, the method comprising:
Loading a sample comprising a mixture of the polypeptide multimers onto a chip comprising a channel having a separation medium effective to act as an obstacle to migration of the polypeptide having the selected disulfide bond pattern; And at least two electrodes are disposed in the channel to induce an electric field,
Applying an electric field across the separation medium of the chip, whereby the polypeptide having the selected disulfide bond pattern is compared to the polypeptide not having the selected disulfide bond pattern. Determining, wherein separation is achieved, and determining the presence of a polypeptide having the selected disulfide bond pattern;
Including the method. 3. The method according to claim 1 or 2, wherein the method further comprises determining the presence of a polypeptide impurity. 2. The method of claim 1, wherein the IgG4 polypeptide having the selected disulfide bond pattern is a half antibody. 3. The method of claim 2, wherein the polypeptide having the selected disulfide is a half antibody. 6. The method of claim 5, wherein the half antibody is of the IgG4 class. 2. The method of claim 1, wherein the IgG4 polypeptide having the selected disulfide bond pattern is produced recombinantly. The method according to claim 1 or 2, wherein the polypeptide is produced recombinantly. The method according to claim 1 or 2, wherein the polypeptide having the selected disulfide bond pattern is produced recombinantly. 2. The method of claim 1, wherein the IgG4 polypeptide that does not have a selected disulfide bond pattern is an anti-integrin antibody. The method of claim 2, wherein the mixture comprises an anti-integrin antibody. The method according to claim 10 or 11, wherein the anti-integrin antibody is produced recombinantly. The method according to claim 1 or 2, wherein the sample is obtained from a growth medium of a cell culture. 3. The method of claim 1 or 2, wherein the sample comprises a polypeptide having a selected disulfide bond pattern of about 1 to about 5000 [mu] g / ml. The method according to claim 1 or 2, wherein the separation medium is a gel polymer. The method of claim 1 or 2, wherein the separation medium is non-reducing. The method according to claim 1 or 2, wherein the movement of the polypeptide is detected using a fluorescence detector. The method of claim 1 or 2, wherein the electric field is non-alternating. The method of claim 1 or 2, wherein the separation further comprises isoelectric focusing. The method according to claim 1 or 2, wherein the separation depends on the molecular weight of the polypeptide. The method of claim 1 or 2, wherein the tip comprises a precast gel polymer. A kit for detecting the presence of a polypeptide having a selected disulfide bond pattern, the kit comprising a chip and instructions for performing the method of claim 1. A kit for determining the purity of a therapeutic polypeptide having a selected disulfide bond pattern, the kit comprising a chip and instructions for performing the method of claim 1. 24. The kit of claim 22 or 23, wherein the kit comprises a separation medium, a non-reducing buffer, a protein dye, a formulation buffer, and a means for inducing an electric field through the separation medium. A kit further comprising a selected component. 24. The kit of claim 22 or 23, wherein the kit further comprises instructions for determining the presence of a polypeptide impurity. 24. The kit of claim 22 or 23, wherein the kit further comprises one or more polypeptide standards.

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